LOCUS       sequence-481608-        6455 bp    DNA     circular SYN 31-JUL-2025
DEFINITION  Expression of eeBxb1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6455)
  TITLE     ONE-STEP tagging: a versatile method for rapid site-specific 
            integration by simultaneous reagent delivery
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6455)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 31, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: EF1a_eeBxb1_Int_2x_NPL_c-term_NLS_pTwist
FEATURES             Location/Qualifiers
     source          1..6455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(1335..1403,1404..2195)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1553..1572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      2208..2344
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2489..2560
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(2565..2611)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    2629..2677
                     /note="synthetic polyadenylation signal"
     promoter        2714..3892
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2945..3883
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3840..3860
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5493..5540
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5550..5597
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5666..5787
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5677..5696)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5731..5750
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      5902..5948
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      5978..6114
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6159..6230
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcattta aatttccgaa
      841 ctctccaagg ccctcgtcgg aaaatcttca aacctttcgt ccgatccatc ttgcaggcta
      901 cctctcgaac gaactatcgc aagtctcttg gccggccttg cgccttggct attgcttggc
      961 agcgcctatc gccaggtatt actccaatcc cgaatatccg agatcgggat cacccgagag
     1021 aagttcaacc tacatcctca atcccgatct atccgagatc cgaggaatat cgaaatcggg
     1081 gcgcgcctgg tgtaccgaga acgatcctct cagtgcgagt ctcgacgatc catatcgttg
     1141 cttggcagtc agccagtcgg aatccagctt gggacccagg aagtccaatc gtcagatatt
     1201 gtactcaagc ctggtcacgg cagcgtaccg atctgtttaa acctagatat tgatagtctg
     1261 atcggtcaac gtataatcga gtcctagctt ttgcaaacat ctatcaagag acaggatcag
     1321 caggaggctt tcgcatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1441 atcagttggg tgcgcgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1501 agagttttcg ccccgaagaa cgctttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1621 ctcagaatga cttggttgag tattcaccag tcacagaaaa gcatcttacg gatggcatga
     1681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1741 ttctgacaac gattggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1861 gtgacaccac gatgcctgta gcaatggcaa caaccttgcg taaactatta actggcgaac
     1921 tacttactct agcttcccgg caacagttga tagactggat ggaggcggat aaagttgcag
     1981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2041 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2161 ctgagatagg tgcctcactg attaagcatt ggtaaccgat tctaggtgca ttggcgcaga
     2221 aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac
     2281 ggatacggct tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc
     2341 ttaagatcgt ttaaactcga ctctggctct atcgaatctc cgtcgtttcg agcttacgcg
     2401 aacagccgtg gcgctcattt gctcgtcggg catcgaatct cgtcagctat cgtcagctta
     2461 cctttttggc agcgatcgcg gctcccgaca tcttggacca ttagctccac aggtatcttc
     2521 ttccctctag tggtcataac agcagcttca gctacctctc aattcaaaaa acccctcaag
     2581 acccgtttag aggccccaag gggttatgct atcaatcgtt gcgttacaca cacaaaaaac
     2641 caacacacat ccatcttcga tggatagcga ttttattatc taactgctga tcgagtgtag
     2701 ccagatctcg tgaggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     2761 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt
     2821 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     2881 gtatataagt gcactagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     2941 acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg
     3001 cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt
     3061 tggaagtggg tgggagagtt cgtggccttg cgcttaagga gccccttcgc ctcgtgcttg
     3121 agttgtggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc
     3181 ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac
     3241 gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatcagcaca ctggtatttc
     3301 ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag
     3361 gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctgcccggcc
     3421 tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc
     3481 ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag
     3541 cacaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa
     3601 aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc
     3661 caggcacctc gattagttct ccagcttttg gagtacgtcg tctttaggtt ggggggaggg
     3721 gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg
     3781 gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct
     3841 caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gaaaactacc
     3901 cctaaaagcc aaaagatctt tgtcgatcct accatccact cgacacaccc gccagcggcc
     3961 gctgccaagc ttccgagctc tcgaattcca ccatgagagc cctggtcgtg attagactga
     4021 gcagagtgac agacgctact acttcccctg agagacagct ggagtcatgc cagcagctgt
     4081 gcgcacagag aggatgggac gtggtcggag tggcagagga cctggacgtg agcggcgctg
     4141 tcgatccatt tgacagaaag cggagaccca acctggcaag gtggctggct ttcgaggaac
     4201 agccctttga tgcgatcgtc gcctacagag tggacaggct gacacgctct attcgacatc
     4261 tgcagcagct ggtgcattgg gccgaggacc acaagaaact ggtggtcagt gcaactgaag
     4321 cccacttcga taccacaact ccttttgccg ctgtggtcat cgcactgatg ggcaccgtgg
     4381 cccagatgga gctggaagct atcaaggagc gaaaccggag tgcagcccat ttcaatattc
     4441 gggccgggaa atacagagga tcactgcccc cttggggcta tctgcctacc cgggtggatg
     4501 gggagtggag actggtgcca gaccccgtcc agagagagag gattctggaa gtgtaccacc
     4561 gggtggtcga taaccacgaa ccactgcatc tggtcgccca cgacctgaat aggcgcggcg
     4621 tgctgagccc aaaagattat tttgctcagc tgcagggaag ggagccacag ggacgaaaat
     4681 ggtccgctac cgccctgaag cggagcatga tcagtgaggc tatgctgggc tatgcaactc
     4741 tgaatgggaa aaccgtccgg gacgatgacg gagcccctct ggtgcgggct gagcctattc
     4801 tgacacgcga gcagctggaa gctctgcggg cagaactggt gaagaccagt agagccaaac
     4861 ctgccgtgag cacaccaagc ctgctgctga gggtgctgtt ctgcgccgtc tgtggggagc
     4921 cagcatacaa gtttgccggc gggggaagaa aacatccccg ctatagatgc cggagcatgg
     4981 gattccctaa gcactgtgga aacggcactg tggctatggc tgagtgggac gccttttgcg
     5041 aggaacaggt gctggatctg ctgggagacg ccgagaggct ggaaaaagtg tgggtcgctg
     5101 gcagcgactc cgctatcgag ctggcagaag tcaatgccga gctggtggat ctgacctccc
     5161 tgatcggatc tcctgcatac agggcaggct ccccacagcg agaagctctg gacgcacgaa
     5221 ttgctgcact ggcagctcga caggaggaac tggagggact ggaagcacga ccttctggat
     5281 gggagtggcg agaaacaggc cagcggtttg gggattggtg gcgcgagcag gacacagcag
     5341 ccaagaacac ttggctgaga agcatgaatg tcaggctgac tttcgatgtg cgcggcgggc
     5401 tgacccgaac cattgatttt ggcgacctgc aggagtatga acagcacctg aggctgggga
     5461 gtgtggtgga gagactgcat actggaatga gcaagagacc agcggcgacg aagaaagcgg
     5521 gccaggcgaa aaaaaagaag ggaagcggca aaaggccggc ggccacgaaa aaggccggcc
     5581 aggcaaaaaa gaaaaagtga ggatccgcag gcctctgcta gcttgactga ctgagataca
     5641 gcgtaccttc agctcacaga catgataaga tacattgatg agtttggaca aaccacaact
     5701 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     5761 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     5821 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatt
     5881 ggcccatctc tatcggtatc gtagcataac cccttggggc ctctaaacgg gtcttgaggg
     5941 gttttttgtg cccctcgggc cggattgcta tctaccggca ttggcgcaga aaaaaatgcc
     6001 tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac ggatacggct
     6061 tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc ttaaggtcgt
     6121 cagctatcct gcaggcgatc tctcgatttc gatcaagaca ttcctttaat ggtcttttct
     6181 ggacaccact aggggtcaga agtagttcat caaactttct tccctcccta atctcattgg
     6241 ttaccttggg ctatcgaaac ttaattaacc agtcaagtca gctacttggc gagatcgact
     6301 tgtctgggtt tcgactacgc tcagaattgc gtcagtcaag ttcgatctgg tccttgctat
     6361 tgcacccgtt ctccgattac gagtttcatt taaatcatgt gagcaaaagg ccagcaaaag
     6421 gccaggaacc gtaaaaaggc cgcgttgctg gcgtt
//
